Structural elucidation of ionized molecules of complex structure, such as proteins is often carried out using a tandem mass spectrometer, where a particular precursor ion is selected at the first stage of analysis or in the first mass analyzer (MS-1), the precursor ions are subjected to fragmentation (e.g., in a collision cell), and the resulting fragment (product) ions are transported for analysis in the second stage or second mass analyzer (MS-2). The method can be extended to provide fragmentation of a selected fragment, and so on, with analysis of the resulting fragments for each generation. This is typically referred to an MSn spectrometry, with n indicating the number of steps of mass analysis and the number of generations of ions. Accordingly, MS2 corresponds to two stages of mass analysis with two generations of ions analyzed (precursor and products). A resulting product spectrum exhibits a set of fragmentation peaks (a fragment set) which, in many instances, may be used as a fingerprint to derive structural information relating to the parent peptide or protein.
Unfortunately, the above-described procedure of sequentially isolating and fragmenting each precursor ion, in turn, may not provide great enough throughput for analyses of complex mixtures of biomolecules. For emerging high-throughput applications such as proteomics, it is important to provide as-yet unattainable speeds of analysis, on the order of hundreds of MS/MS spectra per second. The throughput may be increased by obtaining spectra containing a mixture of fragment sets (a “multiplexed” spectrum), the mixture produced by fragmenting multiple parent ions simultaneously, instead of sequentially. The final multiplexed spectrum contains products from a mixture of precursors, in contrast to an MS/MS spectrum in which the products come from a single isolated precursor.
Procedures for obtaining and analyzing multiplexed spectra can potentially reduce hardware complexity, since an upstream mass analyzer may be eliminated. Analysis of product ions produced by multiple precursor ions can also better utilize the spectral bandwidth of high-resolution mass analyzers, such as Fourier Transform Ion Cyclotron Resonance and Orbitrap mass spectrometers. However, interpretation of the potentially large number of fragment peaks in the resulting multiplexed spectrum can be challenging.
Multiplexing is a general strategy for increasing throughput when the capacity of a communication channel far exceeds what is required to send an individual message at a specified fidelity. Under certain conditions, it may be possible to send multiple messages through the channel simultaneously without appreciable information loss. In communication systems, the individual signals are encoded before being combined at the transceiver to allow the detected signal to be “demultiplexed” or separated into the original component signals at the receiver. The two most common examples of multiplexing are time and frequency multiplexing. In either case, the channel is partitioned into discrete sub-channels.
In the field of mass analysis, the simultaneous measurement of multiple ions by a Fourier transform mass spectrometer (e.g., LTQ-FT or LTQ-Orbitrap) is an example of frequency multiplexing. The signal from each ion populates a narrow band (of fixed width) in the frequency spectrum of the Fourier-transformed transient signal. Typically, these bands are distinct, i.e., non-overlapping, and can be trivially separated. In theory, the channel capacity of a Fourier-transform mass spectrum is the ratio of the spectrum bandwidth divided by the bandwidth of an individual ion signal.
A Fourier transform mass spectrum has sufficient channel capacity to allow the simultaneous measurement of thousands of distinct ion masses, corresponding to neutral molecules present in a sample. However, the “code”, i.e., representing molecules by their masses, is degenerate, since multiple distinct molecules (e.g., isomers) can have identical elemental compositions and therefore identical masses. Furthermore, molecules with masses that are distinct, but differ by less than the nominal mass accuracy, can be misidentified.
To overcome this limitation, additional information about the molecule's identity can be obtained, by breaking the molecule into fragments and measuring the masses of these product ions. The covalent structure of a molecule, which distinguishes it from its isomers, can be inferred from a sufficiently informative MS/MS spectrum and perhaps additional a priori information. Commercially available software products such as MASCOT and SEQUEST have been used to identify peptides by matching a list of masses extracted from such spectra to predicted product ion masses generated from protein sequences stored in proteomic databases. These programs often provide correct identifications even when the product ions are measured with only unit mass accuracy and resolution. Unfortunately, in conventional practice, an entire spectrum is used to measure the product ions from one precursor. This represents a dramatic bottleneck in throughput.
The present invention takes advantage of the concept that the additional information provided by high-mass-accuracy (e.g. 1 part-per-million (ppm) rather than unit mass accuracy) and high-resolving-power measurements of product ions can support mass-spectral de-multiplexing. Such de-multiplexing permits greater sample throughput. In other words, the availability of high-resolution and high-accuracy spectrometers makes it possible, in certain instances, to identify multiple precursor molecules from a single high quality spectrum that contains a mixture of product ions derived by fragmentation of these multiple precursors. The additional mass accuracy of the fragments can enable development of algorithms to discover the correct assignment of product ions to precursors while also compensating for uncertainties, errors, and losses associated with the assignment process.
Such analysis of multiplex MS/MS spectra may make use of existing algorithms, such as MASCOT and SEQUEST to subsequently identify each of the precursors. A preprocessing step would partition product ions from a multiplex spectrum into multiple virtual MS/MS spectra, each of which would contain product ions from only a single precursor. Formation of virtual MS/MS spectra according to the invention thus represents “synthetic isolation” of precursors.
A previously described MS/MS demultiplexing method (PCT International Patent Application Publication WO 2008/003684 A1; inventor, Scigocki) has described the use of “correlation laws” to map pairs, triplets, or arbitrarily large subsets of product ions to a precursor ion. A correlation law essentially states that the masses of the product ions (formed by multiplying each mass-to-charge ratio by an integer representing the unknown charge of the ion) sum to the mass of the precursor ion (also formed by multiplying the mass-to-charge ratio by some integer). However, the observed mass-to-charge ratios contain measurement errors so that a “proximity criterion” is necessary to allow for small deviations from the ideal correlation law. In general, because the charges for the precursors and products are unknown, there could be a large number of correlation laws (planes passing through the space formed by combinations of product mass-to-charge ratios). It is plausible that some of the correlation laws may pass within the tolerance of the observed mass-to-charge ratios of some product ions simply by random chance leading to false assignments of product ions to precursors.
From the foregoing discussion, there is a need in the art for improved methods and apparatus for obtaining and resolving multiplexed tandem mass spectra. The present invention addresses such a need.